DNA barcoding for the identification and authentication of medicinal deer (Cervus sp.) products in China

Deer products from sika deer (Cervus nippon) and red deer (C. elaphus) are considered genuine and used for Traditional Chinese Medicine (TCM) materials in China. Deer has a very high economic and ornamental value, resulting in the formation of a characteristic deer industry in the prescription preparation of traditional Chinese medicine, health food, cosmetics, and other areas of development and utilization. Due to the high demand for deer products, the products are expensive and have limited production, but the legal use of deer is limited to only two species of sika deer and red deer; other wild deer are prohibited from hunting, so there are numerous cases of mixing and adulteration of counterfeit products and so on. There have been many reports that other animal (pig, cow, sheep, etc.) tissues or organs are often used for adulteration and confusion, resulting in poor efficacy of deer traditional medicine and trade fraud in deer products. To authenticate the deer products in a rapid and effective manner, the analysis used 22 deer products (antler, meat, bone, fetus, penis, tail, skin, and wool) that were in the form of blind samples. Total DNA extraction using a modified protocol successfully yielded DNA from the blind samples that was useful for PCR. Three candidate DNA barcoding loci, cox1, Cyt b, and rrn12, were evaluated for their discrimination strength through BLAST and phylogenetic clustering analyses. For the BLAST analysis, the 22 blind samples obtained 100% match identity across the three gene loci tested. It was revealed that 12 blind samples were correctly labeled for their species of origin, while three blind samples that were thought to originate from red deer were identified as C. nippon, and seven blind samples that were thought to originate from sika deer were identified as C. elaphus, Dama dama, and Rangifer tarandus. DNA barcoding analysis showed that all three gene loci were able to distinguish the two Cervus species and to identify the presence of adulterant species. The DNA barcoding technique was able to provide a useful and sensitive approach in identifying the species of origin in deer products.


Introduction
Cervidae, which is more commonly known as the deer family, is the second-most diverse group of ruminants and is widely distributed all over the world [1].At present, a total of 18 genera and more than 40 species have been recorded, with members of Cervus being one of the important genera that has been domesticated and introduced to other nonnative countries as park animals and food sources [2,3].To date, a few countries have established the full domestication of deer for farming, which is commercially important in providing deer meat (venison) to an international market, velvet antler for the oriental medicine trade, and other less important products such as hides, pizzles, ligaments, and teeth [4].
There are at least five species of Cervus that are recorded in China, including C. albirostris (Thorold's deer), C. canadensis (elk), C. elaphus (red deer), C. eldii (Eld's deer), and C. nippon (sika deer) [5].In the past, most of these deer species were used for medicinal purposes; various tissues and organs of deer, including the fetus, penis, tail, heart, meat, blood, and bone, were used in various applications in traditional medicine production, supplementary diet, wine production, etc. [6].Health-benefiting features from deer products were also proven legitimate through scientific approaches [7][8][9].As demand for deer products has seemingly increased over the years, overhunting and the loss of natural habitat due to urbanization have threatened the survival of these species in the wild.To cater to the demand for deer products, the Chinese government has promoted deer farming and stipulated that only farmed red deer and sika deer can be used for food and medicinal purposes; wild deer are generally protected animals by local enforcement agencies, and trades involving these wild deer are prohibited.Consequently, only deer antlers from red deer and sika deer are recognized as sources of traditional Chinese medicine (TCM) [10].Because deer products are expensive and limited, cases of dishonest traders involved in adulterating deer products using body parts from pigs, cattle, and sheep have been reported [11,12].As China is one of the main producers of deer products for both the local and international markets, it is important to ensure that trade fraud involving deer products is resolved due to food safety issues.
In general, identification of the source of origin in deer products greatly relies on the morphological characteristics [13] coupled with microscopic [14] and chemical analyses [15].Although species identification of deer products can be accurate based on these methods, experienced or trained personnel are required to ensure that the identification workflow is properly conducted.To overcome this challenge, simple and straightforward identification of the source of origin of deer products is desired.For this reason, DNA barcoding technology is proposed to be an efficient method to perform species identification [16][17][18].For animal identification, The mitochondrial cytochrome c oxidase subunit I (COI or cox1) gene sequence on the mitochondrial genome is generally considered to be the most useful DNA barcode, followed by other gene sequences such as cytochrome b (Cyt b) and ribosomal RNA 12S (rrn12).All of these loci are useful in distinguishing closely related animal species [19,20].For TCM products, the DNA barcoding technique has been proven useful in the authentication of animal-based medicinal materials, including turtle shells [21], bear bile [22], and buffalo horns [23].Although studies on the potential of DNA barcoding techniques for deer antlers, skin, and meat derived from Cervus have been carried out, in which they are restricted to the use of the cox1 sequences [24][25][26][27], no work has been conducted to evaluate the species discrimination strength of the DNA barcodes on other deer products, such as venison, bone, and fetus.To fill this research gap, we aimed to establish a simple and straightforward method to identify the source of origin of deer products using the DNA barcoding technique.A total of three different candidate DNA barcoding regions, including the cox1, Cyt b, and rrn12 genes, were evaluated for their species discrimination rates based on 22 deer product samples obtained from various sources.The findings of this study will provide an effective means for the trade management of medicinal materials as well as the food safety of deer products in the future.

Sample collection
Sampling was carried out between July and August 2021.For the blind samples, a total of 22 deer products, of which 15 were thought to be derived from sika deer and seven from red deer, were purchased from various deer farms and pharmacies in Hainan Province.These blind samples were in the form of antler, bone, fetus, meat, penis, skin, and tail, in which their species of origin could not be identified through the naked eye.The species identity of each blind sample provided by the sellers was recorded, and they were not informed of the study beforehand.Two identified samples that are in the form of antlers were purchased from the China National Institute for Food and Drug Control (CNIFDC), of which SD-ref is derived from C. nippon (sika deer) and RD-ref is derived from C. elaphus (red deer) and were included as references.All the samples were oven-dried at low temperatures prior to being deposited at the Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences (Table 1).

Total genomic DNA extraction
Before total genomic DNA extraction took place, the samples were surface sterilized with a 70% ethanol solution and cut into smaller sizes.With the help of liquid nitrogen, the samples were pulverized using a medium-flux tissue grinder (Dinghaoyuan Technology, China).Total genomic DNA extraction was carried out using an Omega Tissue DNA Kit D3396 (Omega Bio-technologies, USA) based on the manufacturer's protocol with slight modifications.A total of 100 mg of pulverized sample material was added to a 1000 μL mixture of TL and BL buffers.The incubation time was increased to 6 h, while the subsequent steps followed the manufacturer's protocol.The concentration and purity of the DNA extract were determined using Nanodrop 2000 (Thermo Scientific, USA).
experimental materials were kept in the herbarium of the Analysis and Identification Center of the Hainan Branch of the Institute of Medicinal Plants, Chinese Academy of Medical Sciences.

DNA analysis
The purity (ratio 260/280) of the total genomic DNA extracted from all 22 blind samples was in the acceptable range of 1.8 to 2.0, which indicated good DNA quality (Table 1).The DNA concentrations of the blind samples in the form of meat products, including RD01-05 and SD01-05, were between 800 and 1300 ng/μL; the bone products, including RD06, RD07, SD06, and SD07, were between 1100 and 1300 ng/μL, while other samples, including fetus, penis, antler, and wool, were less than 900 ng/μL.The blind sample that yielded the lowest DNA concentration was derived from a fresh wool specimen, which was 106.9 ng/μL.In addition, the blind samples that were fresh yielded higher DNA concentrations than those that were dried or processed into powder.This could be due to the degradation of DNA when drying or processing treatments were applied.In forensic identification, obtaining quality DNA that enables successful PCR amplification is considered the most crucial step in species identification.Note that DNA recovery in raw food always has a better yield than that in processed food [32].Although successful PCR amplification can be achieved with only a minimum amount of DNA, e.g., 5 ng, which is not visible through the naked eye via gel electrophoresis, yielding sufficient DNA content for PCR amplification could be challenging when involving dry samples, such as hair, bone, and horn/antler [33], which is congruent with our findings.It is believed that the degradation rate of the DNA in dried samples is significant to the storage period; the longer the storage time is, the higher the degradation level.However, oral liquids and granules of Chinese patent medicine are mainly composed of aqueous extracts of TCM.In some cases, TCM pills, including deer product-derived medicines, and a certain amount of honey are usually added as an excipient.Such practices may hinder the DNA extraction and sequencing process [34].Although an optimized DNA extraction protocol could improve the DNA yield from degraded samples, the improvised protocol used in this study exhibited satisfactory results-all blind samples were successfully amplified (100% success rate), suggesting that it should be useful for future applications.

Sequence identity
Upon sequence assembly and after the primer adaptors were trimmed, the sequence lengths of the gene loci cox1, Cyt b, and rrn12 for the two reference samples as well as all the blind samples were 658, 424, and 394 bp, respectively (Table 2).
All the blind samples that were subjected to BLAST analysis returned a match with the record deposited in NCBI GenBank (Table 2).A powerful DNA barcoding locus contains sufficient single nucleotide polymorphisms to tell close species apart [35], while BLAST analysis relies on the sequence similarity of the query sequence to the reference sequence deposited in the GenBank database [36].The sequence alignments for cox1, Cyt b, and rrn12 of the two reference samples resulted in 20, 21, and eight variable sites, respectively (Table 3).With the assumption that the species identities of the reference sequences deposited in the GenBank database are deemed accurate, species identification can be regarded as correctly assigned when all three candidate DNA barcoding loci indicate the same species under a match identity of 100%.Based on such criteria, all the blind samples were indicated with the same species identity when using the three candidate DNA barcoding loci.All 22 blind samples revealed the same species identity through BLAST analysis using the three candidate DNA barcoding loci used, with at least 100% match identity.Based on the BLAST results, we managed to group the 22 blind samples into four different species identities: C. elaphus, C. nippon, D. dama, and R. tarandus.Among the 22 blind samples tested in this study, 11 (RD05-07, SD01-04, SD08, SD13-15) were identified as C. nippon; seven (RD01-04, SD09, SD11-12) were identified as C. elaphus; three (SD05-07) were identified as D. dama; and one (SD10) was identified as R. tarandus.Incongruence between the species identity that was provided by the seller and the result from the BLAST analysis was detected among the ten blind samples tested.Based on the BLAST results, three of the blind samples (SD09, SD11-12) that were derived from red deer (C.elaphus) were misidentified by the seller to originate from sika deer (C.nippon), while four samples (SD05-07, SD10) were adulterated with body parts from fallow deer (D. dama) and reindeer (R. tarandus).And three blind samples (RD01-07) that were thought to originate from red deer (C.elaphus), were identified as sika deer (C.nippon).
The NJ trees based on cox1, Cyt b, and rrn12 analyzed separately revealed that all species were clustered in their respective branches.For DNA barcoding analysis, species delineation is considered to be established based on a selected DNA barcoding locus when all species are clustered under different branches, while individuals of the same species are clustered under the same branch; the genetic distance at the intraspecific level should not exceed that at the interspecific level [16].Thus, it is assured that all three proposed DNA barcoding loci are potentially useful in distinguishing C. elaphus and C. nippon from their adulterants using the DNA barcoding technique.Following the clustering pattern of each NJ tree, it can be concluded that seven blind samples (RD01-04, SD09, SD11-12) were derived from C. elaphus; 11 blind samples (RD05-07, SD01-04, SD08, SD13-15) were derived from C. nippon; three blind samples (SD05-07) were derived from D. dama; and one blind sample (SD10) was derived from R. tarandus.The results from the NJ tree are congruent with those from the BLAST analysis.Based on the three NJ trees, we proposed that the candidate DNA barcoding loci cox1 and Cyt b are suitable for the identification and authentication of deer products derived from C. elaphus and C. nippon.Although weak bootstrap support values are detected in the NJ trees, the branch support value is not considered an important feature in DNA barcoding analysis [16].The candidate DNA barcoding locus, rrn12, was not selected as a potential DNA barcode in this study because the sequence variation between C. nippon and C. canadensis was not sufficient to separate the branches of the two species distinctly (Fig 1 ), making it less advantageous than the other two candidate DNA barcoding loci.An ideal genetic variation in barcoding should be significantly greater between species than within species and form a distinct interval between them, called the barcoding gap [37].As long as the barcoding gap of different species is higher than that of the same species, it is generally considered that this fragment can be used for DNA barcoding identification.It is noteworthy to mention that the   standards obtained from the CNIFDC were correctly identified at the species level; RD-ref and SD-ref were derived from C. elaphus and C. nippon, respectively.To provide a reference for accurate species identification, the sequences for both reference samples were deposited into NCBI GenBank under the accession numbers OP849627 and OP849628 (cox1), OP866991 and OP866992 (Cyt b), and OP856556 and OP856557 (rrn12).Adulteration of food and TCM material has become a growing concern.In recent years, the adulteration of TCM has affected people's health and drug efficacy [38].Deer products from sika deer and red deer are considered authentic in China; however, due to the lack of effective identification means, dishonest traders not only might replace deer products with body parts from adulterant species, but there are cases where authentic deer products were replaced with body parts from other deer species aside from C. elaphus and C. nippon, such as fallow deer, mule deer, reindeer, sambar, etc. [39], as demonstrated in this study for blind samples SD05-07 and SD10.As SD05-07 were purchased from a deer farm, it is possible that the traders or farmers are trying to take advantage of their clients by convincing them that they are actually selling only deer products from C. elaphus and/or C. nippon from their farm.
Species differentiation between D. dama and the two Cervus species has been proposed to be carried out using skeletal or teeth features [40,41] but not meat.At the molecular level, studies to distinguish these taxa are limited to real-time PCR [42] and PCR-RFLP [43] techniques.For real-time PCR, the technique is suitable to detect the presence of DNA from different closely related species in processed/unprocessed meat mixtures [44]; however, a set of reference DNA is required every time the experiment is conducted.Unlike real-time PCR, DNA barcoding utilizes the reference sequences that are deposited in NCBI GenBank for comparison and phylogenetic tree reconstruction.Furthermore, experiments based on the real-time PCR technique are not cost-effective when compared to the DNA barcoding technique [45]; Taq polymerase, fluorescent dye, and qPCR mix are relatively more expensive when compared to conventional PCR mix.For PCR-RFLP, there is a possibility of inconsistency in the results, i.e., differences in banding patterns for the same species due to genetic variation [46].However, the technique is labor intensive and time-consuming, and preknowledge of a suitable endonuclease that could be used for digestion is required to ensure that differences in banding patterns between closely related species can be achieved prior to the experiment [47].A nondestructive method using X-ray microtomography coupled with an SVM classifier was proposed to be useful in differentiating the species of origin for reindeer and red deer antlers [48], in which species differentiation can be carried out by analyzing the alveoli on their antlers.However, such an identification method is known to be useful for prehistoric studies [49], but not in food control due to the need for sophisticated machines and data analyses.Although the consumption of body parts derived from both adulterant species, D. dama and R. tarandus, would not result in any hazardous effects to human health, we speculated that the traders were either confused and misidentified the products beforehand, or the deer products were purposely replaced with those from an adulterant species as a fraudulent and business profit sham.Nevertheless, without a proper farming or handling system, adulterated products cannot be assumed to be safe and free of toxic ingredients at all times; in addition, a decline in medicinal quality is expected [50].Therefore, effective species authentication methods are deemed requisite to ensure the safety and efficacy of TCM, which indirectly provides great significance to the internationalization of the TCM industry.

Conclusion
In this study, the DNA extraction method used is suitable for various forms of deer products, including meat, bones, antlers, etc., some of which were processed into dry or powder form.By using the extracted DNA for subsequent experiments, species identification and authentication of deer products were carried out via BLAST and DNA barcoding analyses.Among the three candidate DNA barcoding loci tested in this study, the gene sequences for cox1 and Cyt b are proposed to be useful for the DNA barcoding of deer products derived from C. elaphus and C. nippon, while the third gene locus, rrn12, exhibited a less advantageous discrimination strength in the DNA barcoding analysis when compared to the other two gene loci.As a result, three blind samples that were labeled as originating from red deer were actually derived from C. nippon, while the presence of adulterant species derived from D. dama and R. tarandus was detected in four blind samples that were labeled as originating from sika deer.Based on the findings of this study, the DNA barcoding technique has been proven to be applicable in species identification and authentication of deer products and could therefore effectively prevent deer product trade fraud and ensure the safe use of authentic deer products.

Table 1 . Information of the body part, form, place of purchase, DNA concentration and quality for the 22 blind samples and two reference samples used in this study. Sample code Label Type Purchase Place DNA conc (ng/μL) Purity 260/280
Note: SD-ref and RD-ref are reference samples; SD stands for Sika deer samples, and RD stands for Red deer samples.https://doi.org/10.1371/journal.pone.0297164.t001